The FOXO-Forkhead transcription factors have important roles in regulation of the cell cycle, apoptosis, and stress responses in mammalian cells; however, little is known about their roles in tumorigenesis. We recently found that (1) nuclear FOXO3a inhibits cell proliferation and tumorigenesis of breast cancer cells; (2) phosphorylation of FOXO3a by I?B kinase-( (IKK() leads to nuclear exclusion of FOXO3a, resulting in increases in cell proliferation and tumorigenesis; and (3) IKK(-mediated phosphorylation of FOXO3a on Ser644 leads to ubiquitination and proteasomal degradation of FOXO3a in the cytoplasm. Thus, IKK( is a key regulator of FOXO3a function. However, regulation of the nucleocytoplasmic shuttling of FOXO3a is not fully understood, and the mechanisms by which FOXO3a promotes apoptosis and tumor suppression are largely unknown. The novel observations that IKK? and IKK? function in the nucleus to regulate NF-?B activity and that IKK( regulates nuclear extrusion of FOXO3a lead us to hypothesize that IKK?, IKK(, and IKK? may have a role in regulating the nucleocytoplasmic shuttling of FOXO3a and its activity in the nucleus, where it regulates the expression of growth-related proteins to elicit its tumor suppressive function. We also recently found that FOXO3a is associated with the ?-transducing repeat-containing protein (?-TRCP) in vivo, suggesting that ?-TRCP is a candidate E3 ubiquitin ligase that mediates the ubiquitination and proteasomal degradation of FOXO3a. Thus, we propose to study the mechanisms underlying the regulation of FOXO3a activity and its role in promoting tumor suppression and to identify novel compounds that activate FOXO activity. The Specific Aims are to (1) investigate the roles of IKK?, IKK(, and IKK? in the regulation of nuclear FOXO3a activity and the role of ?-TRCP ubiquitin ligase in proteolysis of FOXO3a through the ubiquitination- dependent proteasome pathway in tumorigenesis; (2) identify molecular targets of FOXO3a and the regulation of these molecular targets by FOXO3a; and (3) develop a chemical genetic screen for identifying novel small molecules or peptides that can increase the transactivating activity of FOXO factors in breast cancer cells and investigate the roles and effects of these new compounds or peptides in tumor suppression in cell culture and breast cancer animal models. Our goal is to translate our knowledge of FOXO3a -mediated tumor suppression into new anticancer drug discovery for therapeutic intervention. [unreadable] [unreadable]